Benthic anammox and dentrification rates measured in a slurry incubation experiments at four different stations in the Arabian Sea off Pakistan during the METEOR cruise M74/2 in 2007

Vertical distributions of benthic denitrification and anammox rates within the sediment were estimated from slurry incubation experiments. Rates were used to calculate the contribution of anammox and denitrification to the total N-loss. Briefly, MUC sediment cores were sliced in 2 cm intervals and the sediment was diluted and incubated with degassed bottom water in a gas tight bag. After pre-incubating the bags for 2 h, 15N-labeled substrates were injected into the bags and the slurries were thoroughly mixed. Incubations were performed in the dark at in situ temperatures. The N2 isotope ratio (28N2, 29N2, and 30N2) was determined by gas chromatography-isotopic ratio mass spectrometry (VG Optima, Micromass) and calculated according to Kuypers et al. (2005) and Holtappels et al. (2011), respectively.Furthermore, total organic carbon and nitrogen concentrations were measured of core sediment layers corresponding to those used for rate measurements. Concentrations of organic carbon and nitrogen were determined by combustion/gas chromatography (Carlo Erba NA-1500 CNS analyzer) of dried sediment samples after acidification. The same sediment layer were also used to extract nucleic acids. The concentrations of the DNA in the samples were measured spectrophotometrically with a NanoDrop instrument (Thermo Fisher Scientific Inc.). The biomarker functional gene nirS, encoding the cd1-containing nitrite reductase, for both denitrifiers and marine anammox bacteria were quantified with real-time PCR, using the primers cd3aF/R3cd (5'-GTSAACGTSAAGGARACSGG-3' (Michotey et al., 2000)/5'-GASTTCGGRTGSGTCTTGA-3'; Throback et al., 2004) and Scnir372F/Scnir845R (5'-TGTAGCCAGCATTGTAGCGT-3'/5'-TCAAGCCAGACCCATTTGCT-3'; Lam et al., 2009).本研究通过泥浆培养实验（slurry incubation experiments），估算了沉积物内底栖反硝化（benthic denitrification）与厌氧氨氧化（anammox）速率的垂直分布特征，并利用所得速率计算厌氧氨氧化与反硝化对总氮损失（total N-loss）的贡献占比。简言之，将MUC沉积物岩心以2 cm为间隔进行切片，将沉积物稀释后与脱气底层水一同置于气密袋（gas tight bag）中进行孵育。将气密袋预孵育2小时后，向袋内注入15N标记底物（15N-labeled substrates）并充分混匀泥浆体系，全程避光且维持原位温度开展孵育。采用气相色谱-同位素比值质谱仪（gas chromatography-isotopic ratio mass spectrometry，VG Optima型，Micromass公司）测定N₂同位素比值（²⁸N₂、²⁹N₂与³⁰N₂），并分别根据Kuypers等（2005）及Holtappels等（2011）的方法完成计算。此外，针对速率测定所用的沉积物岩心分层样本，测定其总有机碳（total organic carbon）与总氮浓度：有机碳与总氮浓度通过酸化后干燥沉积物样本的燃烧/气相色谱法（combustion/gas chromatography）测定，所用仪器为Carlo Erba NA-1500 CNS元素分析仪（Carlo Erba NA-1500 CNS analyzer）。上述沉积物分层样本同时用于核酸提取，采用分光光度法结合NanoDrop仪器（NanoDrop instrument，Thermo Fisher Scientific Inc.，赛默飞世尔科技公司）测定样本中DNA浓度。针对反硝化菌与海洋厌氧氨氧化菌，采用实时荧光定量PCR（real-time PCR）定量编码含细胞色素cd1（cd1）亚硝酸还原酶的生物标志物功能基因nirS（nirS）；所用引物对包括cd3aF/R3cd（5'-GTSAACGTSAAGGARACSGG-3'，Michotey等，2000；5'-GASTTCGGRTGSGTCTTGA-3'，Throback等，2004）与Scnir372F/Scnir845R（5'-TGTAGCCAGCATTGTAGCGT-3'；5'-TCAAGCCAGACCCATTTGCT-3'，Lam等，2009）。